John Gregory Marshall

Toronto Metropolitan University

There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immuno absorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin, binds to biotinylated macromolecules like proteins, antib...

Phagocytosis is a highly localized and rapid event, requiring the generation of spatially and temporally restricted signals. Because phosphatidylinositol 3-kinase (PI3K) plays an important role in the innate immune response, we studied the generation and distribution of 3' phosphoinositides (3'PIs) in macrophages during the course of phagocytosis....

The MALDI-TOF spectra of peptides from the sera of normal and myocardial infarction patients produced patterns that provided an accurate diagnostic of MI. In myocardial infarction, the spectral pattern originated from the cleavage of complement C3 alpha chain to release the C3f peptide and cleavage of fibrinogen to release peptide A. The fibrinogen...

Cell surface receptors and their associated signaling pathways on the plasma membrane are key targets in understanding cellular responses. However, the isolation and identification of receptor complexes has been elusive. The Fc receptor was captured from the surface of live cells using microbeads coated with the receptor's cognate ligand, gamma glo...

The purpose of this study was to define the role of secretory phospholipase A2 (sPLA2), calcium-independent PLA2, and cytosolic PLA2 (cPLA2) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils. While fMLP induced the release of extracellular sPLA2 activity and AA, 70% of sPLA2 activity remained associated with the cell. Treatmen...

Previous meta-analysis indicated that plasma or serum proteome groups using various experimental conditions detected different peptides from the same plasma proteins, which is strong evidence for the veracity of blood fluid LC-ESI-MS/MS but also evidences that the trypsin digestion step is a key source of variation in plasma proteomics. Agreement b...

Introduction Proteomic analysis of human plasma by LC–ESI–MS/MS has discovered a limited number of new cellular protein biomarkers that may be confirmed by independent biochemical methods. Analysis of COVID-19 plasma has indicated the re-purposing of known biomarkers that might be used as prognostic markers of COVID-19 infection. However, multiple...

Fetal serum supports the immortal growth of mammalian cell lines in culture while adult serum leads to the terminal differentiation and death of cells in culture. Many of the proteins in fetal serum that support the indefinite division and growth of cancerous cell lines remain obscure. The peptides and proteins of fetal versus adult serum were anal...

Background A practical strategy to discover proteins specific to Alzheimer’s dementia (AD) may be to compare the plasma peptides and proteins from patients with dementia to normal controls and patients with neurological conditions like multiple sclerosis or other diseases. The aim was a proof of principle for a method to discover proteins and/or pe...

INTODUCTIONThere is an urgent need for a simple and sensitive method to elucidate the human plasma proteome to find markers of disease, or therapeutic factors. Human plasma proteome may be obtained from tryptic peptides that results from native digestion using commonly available, sensitive and robust analytical instruments such as linear quadrupole...

BackgroundA practical strategy to discover proteins specific to Alzheimer’s dementia (AD) may be to compare the plasma peptides and proteins from patients with dementia to normal controls and patients with neurological conditions like multiple sclerosis or other diseases. The aim was a proof of principle for a method to discover proteins and/or pep...

Background: A practical strategy to discover sepsis specific proteins may be to compare the plasma peptides and proteins from patients in the intensive care unit with and without sepsis. The aim was to discover proteins and/or peptides that show greater observation frequency and/or precursor intensity in sepsis. The endogenous tryptic peptides of...

The alkaline phosphatase–streptavidin enzyme amplification conjugate (APSA) was diluted and quantified to the equivalent of one enzyme molecule injected on column by monitoring the production of excess adenosine from adenosine monophosphate (AMP) using sensitive and selective enzyme-linked mass spectrometric assay. The APSA enzyme conjugate has a m...

The Empirical Statistical Model (ESM) for decoy library searching fused the expected amino acid sequence of 18 non-human protein standards to a human decoy library. The ESM assumed a priori the standards were pure such that only the 18 nominal proteins were true positive, all other proteins were false positive, there was no overlap in the peptides...

Background: There is a need to demonstrate a proof of principle that proteomics has the capacity to analyze plasma from breast cancer versus other diseases and controls in a multisite clinical trial design. The peptides or proteins that show a high observation frequency, and/or precursor intensity, specific to breast cancer plasma might be discove...

Background It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC–ESI–MS/MS to identify, with a linear quadrupole ion trap to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations. Methods A systematic me...

Abstract Background It may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma by using liquid chromatography and tandem mass spectrometry to identify, quantify and compare the peptides cleaved ex vivo from different clinical populations. The endogenous tryptic peptides of ovarian cancer plasma were compared...

A Rabbit myosin standard, like that used to create the empirical statistical model, was randomly and independently sampled by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The rabbit myosin protein standard appeared pure by SDS-PAGE and CBBR staining but showed man...

The proteins identified from endogenous peptides agreed between serum versus plasma, and tryptic versus non-tryptic peptides, collected by C18 alone and analyzed by liquid chromatography electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) including amyloids, apolipoproteins, haptoglobin, complements, fibrinogens, hemopexin, antitryp...

Thirty human EDTA plasma samples from male and female subjects ranging in age from 24 to 74 years were collected on ice, processed ice cold and stored frozen at −80 °C, in liquid nitrogen (LN2), or freeze dried and stored at room temperature in a desiccator (FDRT) or freeze dried and stored at −20 °C for 1 year (FD-20). In a separate experiment, ED...

The tryptic peptides from ice cold versus room temperature plasma were identified by C18 liquid chromatography and micro electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Samples collected on ice showed low levels of endogenous tryptic peptides compared to the same samples incubated at room temperature. Plasma on ice contained peptid...

Background Normal human EDTA plasma samples were collected on ice, processed ice cold, and stored in a freezer at – 80 °C prior to experiments. Plasma test samples from the – 80 °C freezer were thawed on ice or intentionally warmed to room temperature. Methods Protein content was measured by CBBR binding and the release of alcohol soluble amines by...

The alkaline phosphatase-streptavidin (AP-SA) probe released adenosine (∼267.2 Da) from the substrate adenosine monophosphate, where a signal may be detected from as little as 0.5 micro litres of a 0.1 pg/ml dilution of the probe (2.6x10-22 mole). The signal from the AP-SA probe was linear from 1 to 50 pg/ml by monitoring adenosine release at 268 m...

The human monocyte cell line U937 was differentiated into an adherent macrophage phenotype using phorbol ester (PMA) to assay the phagocytosis of oxLDL that may play a role in atherosclerosis. Microbeads were coated with the inflammatory ligand oxLDL to create a novel phagocytosis assay that models the binding of macrophages to oxidized LDL (oxLDL)...

The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulat...

An enzyme-linked immuno-mass spectrometric assay (ELIMSA) with the specific detection probe streptavidin conjugated to alkaline phosphatase catalyzed the production of adenosine from the substrate adenosine monophosphate (AMP) for sensitive quantification of prostate-specific antigen (PSA) by mass spectrometry. Adenosine ionized efficiently and was...

Protein biomarkers offer major benefits for diagnosis and monitoring of disease processes. Recent advances in protein mass spectrometry make it feasible to use this very sensitive technology to detect and quantify proteins in blood. To explore the potential of blood biomarkers, we conducted a thorough review to evaluate the reliability of data in t...

Unlabelled: A new technology termed ELIMSA combines the specificity and enzymatic amplification of Enzyme Linked Immunosorbent assay (ELISA) with the sensitivity and flexibility of mass spectrometry (MS). At present, substrates for the reporter enzymes horseradish peroxidase (HRP) or alkaline phosphatase (AP) yield colored, fluorescent or luminesc...

The inflammatory activation of macrophages by solid aggregates of oxidized low density lipoprotein (oxLDL) in the intima of the arteries is a decisive event on the path to coronary artery disease. However the receptor mechanism by which oxLDL aggregates interact with the macrophages to trigger their phagocytosis is not well understood. The role of...

The Fc receptors belongs to the immunoglobulin superfamily. The ligand of Fc gamma receptor is the immunoglobulin IgG, which triggers the engulfment of foreign molecules coated by antibodies referred to as phagocytosis. Tetraspanins may act as ‘molecular facilitators,’ grouping specific cell-surface proteins and thus increasing the formation and st...

Certain integrin isoforms were shown to be associated with the Fc receptor complex by ligand affinity chromatography in live cells and mass spectrometry. The Fc receptor complex was captured using 2 lm polysterene beads coated in Immunoglobulin G (IgG) and incubated with live human U937 macrophages. The cells were disrupted using a French press and...

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic par...

This unit describes the isolation of activated Fc receptor complexes from RAW 264.7 macrophages using live-cell affinity receptor chromatography (LARC). The Fc receptor complex is activated and captured by IgG-coated microbeads on the surface of live macrophages. After the cells are disrupted, the receptor complexes are isolated by washing and sucr...

The proteins secreted by prostate cancer cells (PC3(AR)6) were separated by strong anion exchange chromatography, digested with trypsin and analyzed by unbiased liquid chromatography tandem mass spectrometry with an ion trap. The spectra were matched to peptides within proteins using a goodness of fit algorithm that showed a low false positive rate...

The Fc receptor complex and its associated phagocytic cytoskeleton machinery were captured from the surface of live cells by IgG coated microbeads and identified by mass spectrometry. The random and independently sampled intensity values of peptides were similar in the control and IgG samples. After log transformation, the parent and fragment inten...

FMS-like tyrosine kinase ligand (FLT3L) is a cytokine responsible for the proliferation and differentiation of hematopoietic progenitor and stem cells. Most notably, the FLT3L-FLT3 receptor complex has been shown to be involved in several hematological malignancies including approximately 45% of acute myeloid leukemia cases, 15% myelodysplastic syn...

The simplest model-that authentic tandem mass spectrometry (MS/MS) spectra are no different from noise, random spectra, or false-positive results-may be directly examined by chi-square comparison of the peptide-to-protein distribution. The peptide-to-protein distribution of a set of 4151 redundant blood proteins identified by X!TANDEM indicated tha...

It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post-translational modifications. However, blood is a complex mixture t...

Many peptides of biological or medicinal importance may be derived from proteolytic actions and are found at low concentrations in human blood fluids. Endogenous polypeptides from human serum were precipitated in acetonitrile and the precipitate was then selectively extracted with water modified by organic solvents and collected over C18 resin. Ext...

Precipitation and selective extraction of human serum endogenous peptides analysis by quadrupole time-of-flight mass spectrometry reveals posttranslational modifications and low-abundance peptides

The endogenous peptides of human serum may have regulatory functions, have been associated with physiological states, and their modifications may reveal some mechanisms of disease. In order to correlate levels of specific peptides with disease alongside internal standards, the polypeptides must first be reliably extracted and identified. Endogenous...

Sequence analysis of the blood peptides and their qualities will be key to understanding the mechanisms that contribute to error in LC-ESI-MS/MS. Analysis of peptides and their proteins at the level of sequences is much more direct and informative than the comparison of disparate accession numbers. A portable database of all blood peptide and prote...

A goodness of fit test may be used to assign tandem mass spectra of peptides to amino acid sequences and to directly calculate the expected probability of mis-identification. The product of the peptide expectation values directly yields the probability that the parent protein has been mis-identified. A relational database could capture the mass spe...

Many proteomics studies are limited to the identification of only the most abundant proteins in a sample due to the high sample complexity in most proteomes. We have here addressed this problem by prefractionation of human blood samples using microchromatography. We show that our approach resulted in high-stringency tryptic peptides identified by L...

The instant invention involves the use of a combination of preparatory steps in conjunction with mass spectroscopy and time-of-flight detection procedures to maximize the diversity of biopolymers which are verifiable within a particular sample. The cohort of biopolymers verified within such a sample is then viewed with reference to their ability to...

The instant invention involves the use of a combination of preparatory steps in conjunction with mass spectroscopy and time-of-flight detection procedures to maximize the diversity of biopolymers which are verifiable within a particular sample. The cohort of biopolymers verified within such a sample is then viewed with reference to their ability to...

The instant invention involves the use of a combination of preparatory steps in conjunction with mass spectroscopy and time-of-flight detection procedures to maximize the diversity of biopolymers which are verifiable within a particular sample. The cohort of biopolymers verified within such a sample is then viewed with reference to their ability to...

Blood peptides can be concentrated, extracted, and analyzed with strong signal-to-noise ratios by precipitation in organic solvents followed by extraction in water. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) hybrid quadrupole time-of-flight (Qq-TOF) were used to analyze the precipitated and extracted endog...

The instant invention involves the use of a combination of preparatory steps in conjunction with mass spectroscopy and time-of-flight detection procedures to maximize the diversity of biopolymers which are verifiable within a particular sample. The cohort of biopolymers verified within such a sample is then viewed with reference to their ability to...

Prostate-specific antigen measurement, widely used for early detection of prostate cancer (CaP), suffers from low specificity. Additional tumor markers are needed for the early detection of clinically relevant CaP. Our objective was to perform a qualitative proteomic analysis of conditioned medium (CM) from the CaP cell line PC3(AR)(6). We used a r...

The instant invention involves the use of a combination of preparatory steps in conjunction with mass spectroscopy and time-of-flight detection procedures to maximize the diversity of biopolymers which are verifiable within a particular sample. The cohort of biopolymers verified within such a sample is then viewed with reference to their ability to...

The instant invention involves the use of a combination of preparatory steps in conjunction with mass spectroscopy and time-of-flight detection procedures to maximize the diversity of biopolymers which are verifiable within a particular sample. The cohort of biopolymers verified within such a sample is then viewed with reference to their ability to...